Abstract
RNA interference (RNAi) is a powerful technique enabling the identification of the genes involved in a certain cellular process. Here, we discuss protocols for microscopy-based RNAi screening in protonemal cells of the moss Physcomitrella patens, an emerging model system for plant cell biology. Our method is characterized by the use of conditional (inducible) RNAi vectors, transgenic moss lines in which the RNAi vector is integrated, and time-lapse fluorescent microscopy. This method allows for effective and efficient screening of >100 genes involved in various cellular processes such as mitotic cell division, organelle distribution, or cell growth.
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Acknowledgements
We are grateful to Mitsuyasu Hasebe, Yuji Hiwatashi, Minoru Kubo, and other former and current Hasebe laboratory members for all moss reagents and valuable information regarding the techniques associated with moss culturing and imaging. We also wish to thank Akiko Tomioka and Momoko Nishina for protocol development and Moé Yamada for reading the manuscript. The moss work in our laboratory is supported by the Human Frontier Science Program, the TORAY Science Foundation, and Grants-in-Aid for Scientific Research (15H01227, 15K14540 and 26711012; MEXT, Japan).
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Miki, T., Nakaoka, Y., Goshima, G. (2016). Live Cell Microscopy-Based RNAi Screening in the Moss Physcomitrella patens . In: Azorsa, D., Arora, S. (eds) High-Throughput RNAi Screening. Methods in Molecular Biology, vol 1470. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6337-9_18
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DOI: https://doi.org/10.1007/978-1-4939-6337-9_18
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